Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae require a signaling pathway that is mediated by the G protein beta-subunit Gpa2p and its coupled receptor Gpr1. Gpr1p is a low affinity glucose receptor that responds to high concentrations of glucose in the environment. Transmission of a signal from Gpr1p to Gpa2p results in phenotypes associated with high levels of intracellular cAMP. We have recently identified KRH1 and KRH2 as genes that encode components of the Gpa2p signaling pathway. We have shown that Krh1p and Krh2p act downstream of adenylyl cyclase to inhibit protein kinase A (PKA) by a process that does not involve production of intracellular cAMP. Activation of Gpa2p is thought to relieve the inhibition of PKA by Krh1p and Krh2p, resulting in high levels of PKA activity. The long-term objectives of this project are: 1) To obtain a complete description of the molecular processes that comprise the Gpa2p signal transduction pathway; 2) To understand the biological function of the Gpa2p pathway in terms of an entire cell population undergoing pseudohyphal growth. The first specific aim of this project is to determine how Krh1p and Krh2p regulate PKA. These studies will investigate whether binding of Krh1p and Krh2p to PKA is direct and whether binding inhibits PKA kinase activity. The second specific aim is to determine whether Krh1p and Krh2p control signaling by affecting the localization of PKA. The third specific aim is to investigate whether the GTP-bound form of Gpa2p blocks the inhibitory function of Krh1p and Krh2p by determining the effects of non-activatable and constitutive alleles of GPA2 on Krh1p and Krh2p function. The fourth specific aim is to test whether Gpa2p is specifically activated in cells at the edge of a growing colony in order to suppress stress and starvation responses and to promote growth and pseudohyphal development. The goal of this aim is to develop a reporter system to allow detection of activated Gpa2p at the level of a whole colony undergoing pseudohyphal growth. These studies will provide new information about G protein-mediated signaling pathways, which are essential for the proper functioning of many physiological processes in humans.